Pluripotent stem cells have the ability to both proliferate continuously in culture and, under appropriate growth conditions, differentiate into lineage restricted cell types representative of all three primary germ layers: endoderm, mesoderm and ectoderm (U.S. Pat. Nos. 5,843,780; 6,200,806; 7,029,913; Shamblott et al., (1998) Proc. Natl. Acad. Sci. USA 95:13726; Takahashi et al., (2007) Cell 131(5):861; Yu et al., (2007) Science 318:5858). Defining appropriate growth conditions for particular lineage restricted cell types will provide virtually an unlimited supply of that cell type for use in research and therapeutic applications.
It would be particularly useful to be able to differentiate pluripotent stem cells into hematopoietic lineage cells. Hematopoietic lineage cells develop from the mesoderm layer and include both white and red blood cells, which constitute the immune and circulatory systems, respectively. An unlimited supply of these cells would provide the tools necessary to more fully understand both the development and functioning of both the immune and circulatory systems. It would also provide insight into strategies for modulating immune responses, both beneficial and harmful.
The immune system provides for an innate or non-specific immune response as well as an adaptive or specific immune response. The adaptive immune response is a long lasting protective response and it is this response most vaccine protocols seek to stimulate. Cellular participants in the adaptive immune response include lymphocytes (T cells and B cells) as well as dendritic cells (DC). T cells and B cells eliminate target pathogens by specifically recognizing antigenic epitopes expressed on the pathogen. T cells have cytotoxic capability that is especially adept at targeting virally infected and tumor cells. B cells secrete antibodies which bind target antigens and activate the complement system facilitating opsonization and lysis of the target. Both responses are characterized as memory responses and thus are protective over a period of time. DC play an important role in initiating the adaptive immune response. They present antigen to the lymphocytes in the context of the appropriate major histocompatibility complex (MHC) and thus provide the initial stimulus for mounting the adaptive immune response. A ready supply of DC could provide a means for generating either a therapeutic or prophylactic immune response in a host.
A number of studies have demonstrated the potential of DC as vehicles for generating an adaptive immune response (see, e.g., Mayordomo et al., (1995) Nature Med 1:1297; Celluzi et al., (1996) J. Exp. Med. 183:283; Su et al., (1998) J. Exp. Med. 188:809) including studies that have investigated the effects of irradiating DC (see, e.g., Cao et al. (2004) Cell Biology International 28:223; Merrick et al., (2005) British Journal Of Cancer 92:1450; Young et al. (1993) Blood 81(11):2987; Denfield et al. (2001) Journal Of Leukocyte Biology 69:548; Dudda et al. (2004) Journal of Investigative Dermatology 122:945).
The potential of dendritic cells along with the promise of pluripotent stem cells have lead several investigators to attempt to differentiate pluripotent stem cells into DC or their precursors (see, e.g. U.S. Pat. No. 7,247,480; U.S. Patent Publication Nos.: 2002/0086005; 2003/0153082; 2006/0275901; 2006/0147432; 2006/0063255; 2006/0147432; Fairchild et al., (2005) International Immunopharmacology 5:13; Tacken et al., (2007) Nature Reviews Immunology 7:790; Senju et al., (2007) Stem Cells 25(11):2720; Sluvkin et al., (2006) J of Immunology 176:2924; Li et al., (2001) Blood 98(2):335; Kaufman et al., (2001) Proc Natl Acad Sci 98(19):10716; Chadwick et al., (2003) Blood 102(3):906; Zhan et al., (2004) Lancet 364:163; Fairchild et al., (2000) Current Biology 10:1515; Kennedy et al., (2007) Blood 109(7):2679; Ng et al., (2005) Blood 106(5):1601; Fehling et al., (2003) Development 130:4217; Lu et al., (2004) Blood 103(11):4134; Zambidis et al., (2005) Blood 106(3):860; Bandi et al., (2008) AIDS Research and Therapy 5:1; Pick et al., (2007) Stem Cells 25:2206).
Many of these investigators relied on stromal cells and/or feeder cells to grow and/or differentiate their stem cells. The use of feeder cells and stromal cells is cumbersome, expensive, time consuming and difficult to scale up. Some of these investigators used animal products such as animal serum in their protocols. Using animal products, however, carries with it the risk of contamination of the cells with zoogenic infectious agents. Still other investigators relied on random or poorly formulated differentiation protocols resulting in unpredictable outcomes and generally low yield of product.
There is a need for hematopoietic lineage cells differentiated from pluripotent stem cells and for methods of producing these cells that is scalable, economical, efficient, reliable, safe, and capable of providing good yield of product. Various embodiments of the invention described herein meet these needs and other needs as well.